Method of producing biological stains and reagents for biochemical determinations using ultrasonic energy

ABSTRACT

A METHOD FOR THE MANUFACTURE OF STAINS OR OTHER MATERIALS OF THE TYPE EMPLOYED TO TREAT BIOLOGICAL SPECIMENS IS DESCRIBED. ULTRASONIC ENERGY, AT A POWER LEVEL SUFFICIENT TO INDUCE CAVITATION, IS APPLIED TO A MECHANICALLY MIXED COMBINATION OF A SOLUTE IN A LIQUID SOLVENT. A REDUCTION IN MANUFACTURING TIME OF APPROXIMATELY 300:1 IS OBTAINED IN MAKING WRIGHT&#39;&#39;S STAIN SOLUTION AND THE RESULTING PRODUCT PENETRATES THE SPECIMAN APPROXIMATELY 60 TIMES FASTER THAN WRIGHT&#39;&#39;S STAIN PRODUCED BY CONVENTIONAL METHODS. VARIOUS TYPES OF ULTRA-FINE DISPERSIONS WHICH MAY BE PRODUCED BY THE METHOD OF THE INVENTION ARE DESCRIBED.

United States Patent 3,553,311 METHOD OF PRODUCING BIOLOGICAL STAINS AND REAGENTS FOR BIO- CHEMICAL DETERMINATIONS USING ULTRASONIC ENERGY Anthony William Smith, 1321 Pyramid Drive, Las Vegas, Nev. 89108 No Drawing. Filed June 30, 1965, Ser. No. 468,608 Int. Cl. Gtlln 1/30 US. Cl. 424-3 5 Claims ABSTRACT OF THE DISCLOSURE Certain natural. and synthetic dyes are used to stain tissues, cells, cell components and other microscopical specimens in order to facilitate the examination of these materials with the aid of a high-powered microscope. These dyes stain the materials and bring out color differences between cell components and their background or between different parts of the substrate. That is, stains are employed Where it is desired to differentiate chromatically one part of a specimen from another. For example, blood smears are usually stained in a methylene blueeosinate dissolved in methanol, of which Wrights stain is a typical example. This stain also requires a phosphate buffer with pH of 6.4.

In its powder form, Wrights stain contains three different types of dyes, namely, an acid dye, a basic dye, and a neutral dye. Glycerine may be optionally added with the alcohol solvent. Normally, the stain solution is permitted to remain on the blood sample for a period of 24 minutes after which the buffer is applied to aid the staining reaction. The buffer is permitted to remain for an additional 2-6 minutes. Both of these periods of time may vary as determined by the manufacture of the dye, the quality of stain, etc. The methyl alcohol (methanol) also serves to fix the blood smear. The three individual components of the stain selectively act on the white and red blood cells, and on the platelets. Following the buffering period, the microscope slide is rinsed with a buffering solution or with distilled water, and then dried and made ready for examination.

Because of the extremely small size of the specimens being treated and because of the high magnification involved in their examination, it is important that the stain be free from coarse particles and/or precipitates which might otherwise obsecure details of the specimen or might inhibit the penetration of the specimen by the dye colorant. For this reason it has been necessary heretofore, to employ extensive mixing and filtering steps in the manufacture of stain solutions in order to assure the ultra-fine dispersion required for treating microscopical specimens. For example, prior methods of manufacturing Wrights stain have involved aging of the dye solution for periods of several months (typically 9 months) during which time the solution is periodically agitated. Even following such time-consuming processing the finished Patented Jan. 5, 1971 product requires that the stain remain on the specimen for 2-4 minutes after which an additional buffering period of 2-6 minutes is required before the specimen may be examined.

The present invention overcomes the above-noted shortcomings in the manufacture and use of Wrights stain. Specifically, by the novel process of the present invention manufacturing time is greatly reduced and the product of the process is superior in that it requires no more than 10 seconds to complete the staining of the specimen. Buffering may consist merely of rinsing the excess stain from the slide. The novel process of the invention involves treating a solution of dye solute and dye solvent with ultrasonic energy at an intensity sufficient to cause cavitation. Holes (gas bubble cavities) can be created in a liquid by high intensity sound waves. When such a cavity collapses, extremely high pressures are produced. This process is called cavitation and is used in the process of the present invention to disperse the dye solute throughout the solvent. Cavitation results in a uniform dispersion of the dye solute throughout the solvent, obviates aging, periodic shaking, and filtering, and results in a solution which is free from coarse particles, and is highly stable, and resistant to precipitation or deterioration in storage.

The invention is not limited to the making of Wrights stain but also is applicable to other stains and biological specimen treating solutions such as cephalin-choelstero-l, flocculation antigen-stable, and STATim Rees-Ecker platelet diluting fluid. The novel process of the invention is useful wherever an ultra-fine, uniform, dispersion of a multiple-component solution is required for the treatment of biological specimens.

'It is, therefore, a principal object of the invention to provide a novel and improved process for the manufacture of biological treatment solutions in which ultrasonic energy is employed to disperse and stabilize the solute component of the solution in the solvent component.

It is another object of the invention to provide a novel and improved process for preparing biological specimen treating solutions wherein the dispersion of a solute in a liquid solvent is affected by means of ultrasonic energy.

Still another object of the invention is to provide a novel and improved process for the manufacture of microscopical stain solutions and biological specimen treatment solutions.

Still another object of the invention is to provide a novel and improved method for obviating the aging and filtering steps heretofore required in the manufacture of Wrights stain.

It is yet another object of the invention to provide a novel and improved method for the manufacture of microscopical stains having greatly reduced processing times.

A general object of the invention is to provide novel and improved specimen treating solutions and methods of manufacture therefor, which overcome disadvantages of previous products and methods heretofore intended to accomplish generally similar purposes.

Other objects of the invention will in part be obvious and will in part appear hereinafter.

Although certain features of the invention may be applied to various microscopical stains and materials used in connection with the staining of microbiological specimens, it will be convenient to describe the invention as applied to the manufacture of an improved Wrights stain. Having gained an understanding of this form of the invention, persons skilled in the art will appreciate the manner in which the principles of the invention may be applied to other specimen treating solutions.

As applied to the manufacture of an improved form of Wrights stain, the process of the invention comprises the steps of mechanically mixing the dye ingredients in methyl alcohol and thereafter treating the solution with ultrasonic energy having a frequency of approximately 42 kilocycles per second (kc.) at an intensity sufficient to induce cavitation in the solution. The solution is treated for approximately 30 minutes.

There follows a specific example of the process of the invention as applied to the manufacture of Wrights stain solution:

EXAMPLE 1 Add 3.0 grams commercially prepared Wrights stain powder and 30 millilitres of glycerine to 1 litre of acetonefree methyl alcohol. Mechanically mix these ingredients by shaking, stirring, etc. for minutes. Thereafter, irradiate the mixture with ultrasonic energy at a frequency of 42 kc. and a power density of 6.5 watts per square centimetre for 20-30 minutes.

The stain solution prepared in accordance with Example 1 is superior to Wrights stain solution produced by prior methods since it permits immediate use. It is especially important to note that prior methods of preparing a Wrights stain solution require many months of aging, during which time periodic shaking is required. The application of ultrasonic energy to the process results in more than a mere improvement in mixing, as would be expected, since the utilization of ultrasonic energy at the frequency, the power level, and the duration indicated results in an unexpected change in the molecular distribution of the dye solutes in the solvent. A reduction of manufacturing time from 6 to 12 months, to approximately 30 minutes is so marked as to be wholly unexpected to one having ordinary skill in the art. Furthermore, the ultrafine dispersion produced in accordance with this method permits immediate penetration of the dye into the specimen. Heretofore, it has been necessary to allow 2 to 4 minutes for the stain to penetrate the specimen. Buffering with a standard buffer of 6.4-7.0 pH, or distilled water, etc., following the application of the stain produced in accordance with Example 1, results in an excellent staaining of the blood cells. Also, the platelets in the stained smear obtain a more intense coloration.

The solution produced in accordance with Example 1 permits staining by the conventional rack methods involving the following steps:

(1) Apply stain to the slide and immediately add buffer.

(2) Permit stain and buffer to remain on slide for approximately seconds.

(3) Wash.

(4 Dry.

Alternatively, a large number of slides (e.g., 50-100 slides) simultaneously may be clipped in the stain solution for 2-4 seconds after which they are dipped into the buffer solution for 3l0 seconds, then washed and dried. Either of the above procedures will result in excellent staining of the blood cells. Consistent results will be obtained With a very substantial reduction in time as compared with prior solutions and techniques.

While the frequency of the ultrasonic energy specified in Example 1 has been found to be both convenient and satisfactory, it should be understood that the invention need not be limited to such frequency but may employ frequencies over a considerably wider range, as for ex ample, a range from 4 kc. to 400 kc. It has been found that lower frequencies require longer periods of irradiation to accomplish the intended result, and that heating becomes greater with an increase of frequency (above about 300 kc.) because of increased adsorption.

The power level required is that sufiicient to result in some form of cavitation. Cavitation is a generic term applied to a number of ultrasonic effects characterized by the formation and collapse of bubbles in a liquid. The onset of cavitation is influenced by all of the characteristics of the system, i.e. temporary hydrostatic pressure,

type of excitation, viscosity, etc. While there are several types of cavitation including gas-filled and vapor-filled bubbles or cavities 'in the liquid, the present invention always requires that some form of cavitation occur.

It has been found that the workable power range for the process of the present invention is from approximately 1 watt per square centimeter to 25 watts per square centimeter. In a typical practical case, a power level of 6.5 watts per cm. has been found to provide satisfactory results.

As has been mentioned previously, the invention is not limited to the manufacture of Wrights stain solution, but may be applied to other stains and solutions for treatment of biological specimens requiring ultra-fine and highly stable dispersion of a solute in a solvent. Dyes used in staining biological specimens are broadly divided into acid dyes and basic dyes which color the nucleus and other cellular components. Each class further contains dyes which attach directly (direct dyes) and those which attach to an intermediary (mordant) which is either ap plied before, or in the same solution as, the dye. The process of the invention may be applied to all such classes of dyes which are to be dissolved in a solvent.

Orcein, safranin, methylene blue, and crystal violet are typical direct nuclear dyes while carmine, hematoxylin, and celestin blue B are commonly used with mordants. All of these dyes may be dispersed and dissolved in a solvent by the application of ultrasonic energy in accordance with the invention. The various eosins, orange G, ponceau 2R, light green SF, and methyl blue are among the better known basic dyes.

Many basic and acidic dyes form compounds known as neutral dyes which may be used to stain both nucleus and cytoplasm from a single solution. The numerous com pounds of various eosins with various thiazins, such as eosin Y-methylene blue used to stain blood films, are in this category. Other dyes susceptible to the process of the present invention are Mallory-Azan, Feulgen-fast green, azocarmine, and La Cours acetocarmine.

Basic dyes are commonly applied from simple solutions in water or weakethanol. Acid dyes, however, usually require compounding into staining solutions, sometimes of considerable complexity and usually of an empirical composition established by tradition. All except orcein, and some oxazines, must be used indirectly; that is, the object is first impregnated with the dye solution and then differentiated, usually with a weak acid, until the dye is removed from the cytoplasm. After this treatment, hematoxylin, which is pink in acid, must be blued by treatment with a weak alkali.

From the foregoing discussion of dyes used in the composition of specimen treating stains it will be appreciated that the dye solvent may comprise water, methanol, ethanol, solutions of weak acids or bases, or mixtures of these various liquids. Also, the solute material to be dissolved may be other than a dye. For example, it may be desired to treat a biological specimen with an antigen test solution. There follows an example of the application of the process of the invention to the processing of cephalen-cholesterol flocculation antigen-stable.

EXAMPLE 2 Mechanically mix the following until it appears to be uniformly distributed:

105.0 millilitres 1.0% saline solution 15.0 millilitres thimerosal 1:1000 30.0 millilitres prepared antigen emulsion Thereafter irradiate with ultrasonic energy at a frequency of 42 kc. at a power level of 6.5 watts per cm. for 40 minutes.

The test antigen prepared in accordance with Example 2 results in significantly greater dispersion of the particles in the saline solution and also results in a more uniform suspension due to the smaller size of the particles. When a serum of high gamma globulin content (above normal) is added to the test antigen, a more uniform aggregate or clumping will result. This significantly improves the consistency and accuracy of measurement whether obtained by visual or instrumentation techniques.

Standardization of a turbidimetric reading may easily be made by calibrating the supernatent fluid after centrifugation, and thereby obtain quantitative results, if desired.

EXAMPLE 3 STATim Rees-Ecker platelet diluting fluid Mechanically mix the following:

0.75 gram brilliant cresyl blue powder 38.0 grams sodium citrate (reagent grade) 0.1 gram powdered thimerosal 1 litre distilled water Irradiate the mixture with ultrasonic energy at 42 kc., at a power level of 6.5 watts per cm. for 30-40 minutes.

The above formulation contains only about the amount of cresyl blue powder as the conventional formula employed heretofore, yet the solution obtained from the process of Example 3 results in a superior stain. That is, the particles of stain penetrate the platelet immediately. Also, the surrounding background is significantly lighter and the platelets are more intensely stained due to the increased index of penetration. This results in an increase in the ease and accuracy of counting. Prior techniques required that the diluted cells be placed in a moist chamber and allowed to settle and stain for approximately minutes. The stain produced by the method of Example 3 permits the platelets to be stained within a few seconds and the platelet count may be performed as soon as the cells settle. Usually this is about 3-5 minutes. The merthiolate included in the formulation of Example 3 inhibits bacterial growth, and is used in lieu of a formaldehyde solution as employed in formulations of the prior art.

EXAMPLE 4 Solution of idonitrophenol tetrazolium (molecular weight 505.70)

Prepare a l micromole solution of iodonitrophenol tetrazolium (weight/volume) in a 1% solution of Triton X-100 in distilled water, by mechanically mixing. I

Irradiate with ultrasonic energy at 42 kc. at a power level of 6.5 watts per square centimeter for 45 minutes. Following irradiation the solution is ready for immediate use.

Triton X-100 is a solvent manufactured by Z. D. Gilman Inc., Washington 4, DC. Prior methods for the manufacture of iodonitrophenol tetrazolium dye solutions required that the solution be heated for 24 hours at 60 degrees centigrade in order to efiect solution of the dye. Furthermore, solutions prepared by prior techniques only had a storage life of about 60 days when refrigerated; However, the iodonitrophenol tetrazolium solution prepared by the present method may be stored at room temperature for periods in excess of eight months.

It will be evident to those versed in the art, from a consideration of Examples 1-4, how the novel process of, the invention may be extended to other biological stains. For example, Kinyons stain for acid-fast bacilli, prepared in accordance with the standard formula procedure, and thereafter irradiated with ultrasonic energy at a level suflicient to induce cavitation will result in a stain solution which will remain stable indefinitely. The time required to stain a specimen for acid-fast bacilli has, prior to the present invention, required approximately 15 minutes. When prepared in accordance with the method of the present invention the time for staining is reduced to about 2 minutes and results in a desirably more intense staining. I i

It should also be noted, upon comparing the formulations of the Examples 1- 4 with similar solutions of the prior art, that smaller quantities of the dye or other solute are required to accomplish the desired result, due to the greatly reduced particle size which gives a higher index of penetration when the solution is applied to a biological specimen.

Since certain changes may be made in the above process and compositions without departing from the scope of the invention herein involved, it is intended that all matter contained in the above description shall be interpreted as illustrative and not in a limiting sense. Therefore, the invention is to be limited only as indicated by the scope of the following claims.

What is claimed is:

1. In a method for manufacturing a stain of the type used to stain biological specimens and which comprises a dye solute dispersed in a liquid solvent, the improvement comprising:

irradiating a mixture of said dye solute and said liquid solvent with ultrasonic energy having a frequency within the range of 4 kilocycles per second to 400 kilocycles per second and at a power level within the range from 1 watt per square centimeter to 25 watts per square centimeter to cause cavitation in said mixture for a period within the range from 1 minute to 60 minutes.

2. The method of stabilizing a solution of Wrights stain and accelerating the aging thereof, comprising the step of:

irradiating said solution with ultrasonic energy having a frequency in the range of 4 kilocycles per second to 400 kilocycles per second and a power level within the range from 1 watt per square centimeter to 25 watts per square centimeter to induce cavitation therein for a period within the range of one minute to one hour.

3. The method for preparing Wrights stain which comprises:

mechanically mixing Wrights biological stain powder in a solution of methanol and glycerine until said powder is dissolved; and

thereafter irradiating the mixture with ultrasonic energy having a frequency in the range of 4 kilocycles per second and 400 kilocycles per second and a power level within the range from 1 water per square centimeter to 25 watts per square centimeter to induce cavitation therein for a period within the range of one minute to one hour to prevent any significant precipitation in said mixture.

4. The method for preparing cephalin-cholesterol flocculation antigen-stable which comprises:

preparing a mixture consisting essentially of the following components, based on parts by volume:

1 /2 parts thimerosal solution 1:1000

3 parts prepared antigen emulsion 10 /2 parts 1% saline solution thereafter irradiating the mixture with ultrasonic energy having a frequency in the range of 4 kilocycles per second to 400 kilocycles per second and a power level within the range from 1 watt per square centimeter to 25 watts per square centimeter to induce cavitation therein for a period within the range of one minute to one hour to prevent significant precipitation therein upon termination of the irradiation.

5. The method for preparing STATim-Rees-Ecker platelet diluting fluid which comprises:

preparing a mixture consisting essentially of the following components based on parts by weight:

0.75 parts Brilliant Cresyl Blue powder 38.0 parts sodium citrate 0.1 thimerosal powder 100.0 distilled water thereafter irradiating the mixture with ultrasonic energy having a frequency in the range of 4 kilocycles per second to 400 kilocycles and a power level within the range from 1 watt per square centimeter to 25 7 watts per square centimeter to induce cavitation Todd, Clin. Diag., by Lab. Methods, W. B. Saunders, therein for a period within the range of one minute Philadelphia, 1941, pp. 245, 246. to one hour to prevent significant precipitation ilpon Difco Manual, Difco Lab., Detroit, 9th ed. 1953, pp. termination of the irradiation. 324-325.

5 Yeager, J. Soc. Cos. Chem. vol. 8, May 1957, pp. References Cited 139-143, 151-154. FOREIGN PATENTS ALBER 1,025,703 3/1958 Germany 424-3 D M 25 1 52 1 i z g Exafmmer OTHER REFERENCES 10 S515 an Xammer Gammack, Biochim Biophys Acta, vol. 84, 1964, pp. US. Cl. X.R. 576-585. 424-7, 12

Staining Proc., Bio. Stain Comm., Williams & Wilkins, Baltimore, 2nd ed., 1960, pp. 133, 134. 

